Urokinase derivatives covalently bound to fibrinogen

ABSTRACT

Urokinase derivatives comprise urokinase covalently bonded with fibrinogen or urokinase covalently bonded with fibrinogen through an aliphatic diamine and correspond to the general formula: ##STR1## wherein P is fibrinogen, E is urokinase, R is absent or stands for ##STR2## wherein n is 1-12, with a molecular mass of from 360,000 to 440,000 D, a content of protein of 10 to 30% by mass, an esterase catalytic activity of 30-60%. 
     These derivatives have an increased affinity to fibrin and feature a prolonged fibrinolytic effect.

FIELD OF THE INVENTION

The present invention relates to bio-organic chemistry and, moreparticularly, to novel urokinase derivatives possessing affinity forthrombus material and displaying a thrombolytic activity.

BACKGROUND OF THE INVENTION

Known in the art are various urokinase derivatives, for example,water-soluble urokinase derivatives suitable for therapeutic purposescomprising complexes of the enzyme of urokinase with heparin or dextransulphate (cf. U.S. Pat. No. 4,106,992; 1978; Japanese Pat. No. 159387,1977).

These urokinase derivatives are, however, unstable.

When penetrating into blood circulation, they cause destruction ofelectrostatic interactions with the formation of substantially a mixtureof urokinase with the vehicle, which is accompanied by a rapidinactivation of the enzyme and discontinuation of the thrombolyticeffect.

Also known in the art are urokinase derivatives comprising urokinasecovalently bonded with dextran (cf. Japanese Pat. No. 54-113488, 1979).

These derivatives are water-soluble and feature an enhanced stability.However, such modification of the enzyme does not ensure its affinityfor the thrombus material, wherefore an enhanced fibrinolytic activityof blood with the use of such derivatives still does not guarantee aneffective process of thrombolysis.

DISCLOSURE OF THE INVENTION

The present invention is directed to novel urokinase derivativesfeaturing an increased affinity for fibrin and possessing a prolongedfibrinolytic effect owing to a higher stability thereof.

The urokinase derivatives according to the present invention are noveland hitherto unknown in the literature.

The derivatives of urokinase according to the present invention compriseurokinase covalently bonded with fibrinogen, or urokinase covalentlybonded, through an aliphatic diamine, with fibrinogen and correspondingto the general formula: ##STR3## wherein P is fibrinogen, E isurokinase, R is either absent or stands for ##STR4## where n is 1 to 12;with a molecular mass of 360,000-440,000D, protein content of 10-30% bymass, and an esterase catalytic activity of 30 to 60%.

The compounds according to the present invention comprise a product ofbonding of urokinase to fibrinogen which is employed as a vehicle forimmobilization of urokinase.

Owing to proper selection of a matrix of a high-molecular vehicle(fibrinogen) the compounds according to the present invention have anincreased tropism to the thrombus material. This ensures an effectiveprogress of the thrombolytic process. At the same time, a preliminarymodification of fibrinogen with aliphatic diamines hinders destructionof the protein matrix in the course of fibrinolysis. The addition ofurokinase of fibronogen imparts an increased stability to the compoundswhich can also ensure prolongation of the fibrinolytic activity of suchderivatives. As regards their kinetic parameters, the compoundsaccording to the present invention are close to the native enzyme and intheir fibrinolytic effect they are nearly two times more effective ascompared to the starting urokinase.

DETAILED DESCRIPTION OF THE INVENTION

Properties of the novel compounds according to the present inventionhave been studied in in vitro experiments.

The experiment was carried out in parallel with the use of nativeurokinase and the following urokinase derivatives according to thepresent invention: compound I (urokinase attached to fibrinogen by meansof 1,12-dodecamethylenediamine), compound 2 (urokinase added tofibrinogen by means of 1.10-decamethylenediamine), compound 3 (urokinaseadded to fibrinogen by means of 1,7-heptamethylenediamine), compound 4(urokinase added to fibrinogen by means of 1,4-tetramethylene diamine),compound 5 (urokinase added to fibrinogen).

The study of kinetic parameters of an enzymatic hydrolysis of methylether of acetylglycyl lysine (AGLMe) by the compounds according to thepresent invention has shown that these compounds are very close to thenative enzyme (urokinase). The data of the experiment are shown in Table1 hereinbelow.

The AGLMe concentration is 10⁻³ to 10⁻² M, the enzyme concentration inthe cell is 100 IU/ml.

As is seen from the data of Table 1, the best kinetic parameters of theurokinase derivatives according to the present invention are inherent incompound 1 (urokinase attached to fibrinogen by means of1,12-dodecamethylenediamine). The use of the longest aliphatic diamine(the longest "leg") out of the available range of aliphatic diaminesmakes it possible to produce preparations having properties mostresembling those of the native enzyme.

The long "leg" presumably eliminates to a considerable extent thenegative effect provided on the enzyme globule by the matrix which mightbe revealed in a change of the conformation state of the combined enzymemolecule, in steric hindrances emerging upon the interaction of theenzyme with the substrate, and so forth.

                                      TABLE 1                                     __________________________________________________________________________                           Urokinase                                                                            Urokinase de-                                                  Urokinase de-                                                                         derivative                                                                           rivative of                                                                          Urokinase de-                                           rivative of                                                                           of fibrino-                                                                          fibrinogen                                                                           rivative of                                                                           Urokinase de-                                   fibronogen and                                                                        gen and 1.10-                                                                        and 1,7-hepta-                                                                       fibrinogen and                                                                        rivative of                      Kinetic        1,12-dodecame-                                                                        decamethyl-                                                                          methylenedia-                                                                        1,4-tetramethyl-                                                                      fibrino-                         paramet-       thylenediamine                                                                        enediamine                                                                           mine (com-                                                                           diamine (com-                                                                         gen                              ers   Native urokinase                                                                       (compound 1)                                                                          (compound 2)                                                                         pound 3)                                                                             pound 4)                                                                              (Compound 5)                     1     2        3       4      5      6       7                                __________________________________________________________________________    K.sub.M, M                                                                           3.0 × 10.sup.-4                                                                 11.0 × 10.sup.-4                                                                13.0 × 10.sup.-4                                                               14.0 × 10.sup.-4                                                               15.0 × 10.sup.-4                                                                18.0 × 10.sup.-4           k.sub.cat, sec.sup.-1                                                               62       33      30     29     27      21                               __________________________________________________________________________

This is proven by the comparison of kinetic characteristics of nativeurokinase and products of modification thereof. Thus, with shortening"leg" length values of K_(M) for urokinase derivatives are increased andvalues of k_(cat) are decreased. For compound 5 (urokinase combined withfibrinogen without a diamine) a maximum value of K_(M) and a minimumvalue of k_(cat) are characteristic which demonstrates a noticeablereduction in the activity of this derivative in comparison with nativeurokinase.

The addition of urokinase to fibrinogen shifts the pH-optimum of theenzyme catalytical activity relative to hydrolysis of the AGLMe towardsthe acidic side. Thus, the pH-optimum of native urokinase is 8.3, thepH-optimum of the urokinase derivative according to the presentinvention (compound 1) is 7.5. This is explained by elimination of thenegative charge of carboxy groups in a globule of urokinase uponactivation thereof with carbodiimide. The alteration of the residualcatalytical activity of urokinase upon its modification occurs in thefollowing manner: enzyme activity prior to modification--100%, after thestage of carbodiimide activation--88%, after combining with fibrinogenmodified with 1,12-dodecamethylenediamine--57%, after separation of theresulting derivative by the utrafiltration method on an "Amicon"instrument is 52%. The combining of the enzyme with the polymetricmatrix of fibrinogen makes it possible to retain, for the enzymeglobule, a certain mobility (changes of K_(M), k_(cat)) and, inaddition, it imparts an increased stability to the resulting biocatalystcompound which is demonstrated by the data of the following Table 2.

                  TABLE 2                                                         ______________________________________                                                Residual catalytic activity retained by                                       the preparation upon incubation at 50° C.                              in a 0.1M phosphate buffer with pH = 7.5                                      after:                                                                Compounds 3 hours 7 hours 14 hours                                                                             24 hours                                                                             48 hours                              ______________________________________                                        Native uroki-                                                                           80      56      38     10     --                                    nase                                                                          Compound 1                                                                              85      75      60     47     32                                    Compound 2                                                                              86      75      56     49     37                                    Compound 3                                                                              86      73      58     45     40                                    Compound 4                                                                              88      76      60     52     42                                    Compound 5                                                                              90      78      66     56     44                                    ______________________________________                                    

The determination of the residual catalytic activity was carried out ina pH-stat by hydrolysis of a 10⁻³ M solution of AGLMe in a 0.1M KCl atthe pH=7.5 and at room temperature.

The experimental determination of the thrombolytic activity of compoundsof native urokinase and the derivatives of urokinase according to thepresent invention was effected according to the procedure described inthe USSR Inventor's Certificate No. 824023 with the addition, to thewashing solution, of 0.33 mg/ml of human plasminogen. To 0.5 ml of asolution (10 mg/ml) of fibrinogen 0.2 ml of a solution (4 mg/ml) ofthrombin is added and this mixture is maintained for one hour at roomtemperature; during this time the fibrin skeleton of the thrombus isformed. It is placed onto a permeable film into a secured plastic tubewith a diameter of 1.0-1.5 cm with its bottom washed by 15 ml of asolution of a 0.15M phosphate buffer with the pH of 7.4. In thissolution plasminogen is also dissolved (0.33 mg/ml; control) andpreparations of native urokinase and derivatives of urokinase accordingto the present invention which are used in a ratio ensuring theirsimilar esterase (AGLMe) catalytic activity. The dissolution of thefibrin clot is controlled by an increase in the optical density (atλ=280 nm) of the washin solution with time. The results of theexperiments are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                                               Compound of the present in-                                                   vention (urokinase - 1,12-                                                    dodecamethylenediamine-                                Time,  Native urokinase                                                                              fibrinogen)                                            min    A.sup.280                                                                             ΔA.sup.280                                                                       %    A.sup.280                                                                            ΔA.sup.280                                                                     %                                  ______________________________________                                         0     0.292   0         0   0.842  0       0                                  30    0.326   0.034    11   0.879  0.037  12                                  90    0.383   0.091    30   0.971  0.129  43                                 150                          1.083  0.241  80                                 180    0.418   0.126    42   1.144  0.302  100                                240    0.484   0.192    64                                                    270    0.539   0.247    82                                                    300    0.592   0.300    100                                                   ______________________________________                                    

The ratio of the increment of the optical density of the solution (as aresult of destruction of the insoluble fibrin clot and passing of itsfragments into solution upon thrombolysis) to the time period duringwhich it occurred characterizes the speed of the thrombolysis process insuch a system (tgα). In a control experiment no considerable lysis of afibrin clot is observed. For preparations of native urokinase and thederivatives of urokinase according to the present invention (compound 1)the values of tgα are equal to 0.3 and 0.5 respectively which points toa high thrombolytic activity of the derivative of urokinase according tothe present invention and it is also proven by the values of duration ofa complete dissolution of a thrombus equal to 5 and 3 hoursrespectively.

The final results of the experiments on thrombolysis are shown in Table4 hereinbelow.

                  TABLE 4                                                         ______________________________________                                                                  Time of complete                                                  Speed of disso-                                                                           dissolution of                                                    lution of fib-                                                                            fibrin clot,                                        Preparation   rin clot    hours                                               ______________________________________                                        Native urokinase                                                                            0.3         5                                                   Compound 1    0.5         3                                                   Compound 2    0.43        4 hrs 45 min                                        Compound 3    0.40        4                                                   Compound 4    0.36        4 hrs 20 min                                        Compound 5    0.25        5.5                                                 ______________________________________                                    

The data shown in Table 5 demonstrate that with the growth of the "leg"length of the employed diamine the efficiency of the thrombolytic actionof the urokinase derivatives according to present invention isincreased. It might be suggested that the reason of this phenomenonresides in a more favourable ratio, for such derivatives, of the enzymemobility, its distance from the vehicle matrix and its ability of beingbuilt in the thrombus material. It is likely that a further increase ofthe "leg" length is inadvisable, since the stabilizing effect of thevehicle is lost and modification thereof by such bifunctional reagentswith increasing hydrophobicity is hindered due to shortening of theiractual length (twisting of the methylene chain). As experimental datashow, 12 methylene groups in the reagent chain is the length of anintermolecular cross-linking which is close to the optimal one.

The experimental verification of the enhanced tropism of the preparationhas been effected in the following manner: formed clots of fibrin werewashed for a specified period of time (15 minutes) with solutions ofpreparations of native urokinase and the derivatives of urokinaseaccording to the present invention. Then the clots were transfered intosolutions of the same buffer without the enzyme and incubated for threehours. In these solutions the growth of optical density was determined,i.e. the speed of dissolution of the fibrin clot; as the control use wasmade of a buffer containing plasminogen and washing the fibrin clotduring the entire duration of the experiment. It has been shown thatthere occurs a rapid building-in of fibrinogen from the washing solutioninto the fibrin clot. This inclusion is detected visually already within5-15 minutes. The results of the experiments are shown in Table 5hereinbelow.

It follows from the data of Table 5 that the effect of the nativepreparation of urokinase (tgα==0.15) as regards the efficiency ofthrombolysis, is close to the data of the control experiment(tgα==0.14). Fibrinolysis in the control is due to the presence ofplasmin impurity in the plasminogen preparation. A considerably higherthrombolytic activity is revealed by the urokinase derivative--compound1 (tgα=0.3). It means that this very derivative of urokinase has anaffinity towards the fibrin clot, since it is included thereinto uponthe first 15-minutes' incubation and transfered along with the clot intothe buffer solution, wherein the fibrinolytic action of the preparationlasts for 3 hours.

                  TABLE 5                                                         ______________________________________                                               Speed of dissolution of fibrin clot with-                                     in 3 hours in a buffer solution after                                         incubation with the preparation during:                                Preparation                                                                            15 min   15 min  15 min  15 min                                                                              15 min                                ______________________________________                                        Native uroki-                                                                          0.15     0.17    0.14    0.16  0.14                                  nase                                                                          Compound 1                                                                             0.3      0.38    0.42    0.44  0.46                                  Compound 2                                                                             0.22     0.26    0.28    0.30  0.31                                  Compound 3                                                                             0.19     0.22    0.24    0.25  0.27                                  Compound 4                                                                             0.16     0.20    0.23    0.25  0.26                                  Compound 5                                                                             0.11     0.12    0.12    0.14  0.15                                  Control  0.14     0.14    0.14    0.14  0.14                                  ______________________________________                                    

Moreover, upon repetition of the experiment with the same fibrin clotsfor several times (up to 5) it has been observed that the urokinasederivatives according to the present invention have the ability of beingadditionally built into an already lyzed thrombus which is proven byincreasing values of tgα (from 0.3 to 0.46)--characteristic indicator ofthe thrombolysis efficiency. As it is seen from Table 5, a similarability is not inherent in native urokinase.

The process for producing the urokinase derivatives according to thepresent invention is performed in a conventional manner.

The process is carried out in three stages.

(I) Activation of carboxy groups of fibrinogen by means of acarbodiimide and addition of an aliphatic amine thereto.

This process is performed by a conventional technique.

To a solution of fibrinogen in distilled water (pH=3.8) a 100-fold molarexcess of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is added and themixture is incubated for 10 minutes at room temperature, whereafter itis added with a 100-fold molar excess of an aliphatic amine in aphosphate buffer (pH=8.3) at the temperature of 4° C. and pH=4.5 and theincubation is continued for 5 hours. The resulting product (product 1)is dialyzed against distilled water at 4° C., then against the phosphatebuffer at pH=8.3.

(2) Activation of carboxy groups of urokinase by means of acarbodiimide.

To a soution of urokinase preliminarily dialyzed against distilled waterand a phosphate buffer (pH=8.3)1-ethyl-3-(dimethylaminopropyl)carbodiimide is added in the molar ratioof 1:100 and incubation is effected at the temperature of 4° C. for 20minutes (product II).

(3) Addition of urokinase immobilized with respect to fibrinogen.

The reaction of combining of urokinase and fibrinogen is effected bymixing products I and II, followed by incubation of the mixture for 18hours at the temperature of 4° C. Then the resulting preparation ofurokinase covalently bonded with fibrinogen is recovered by means ofgel-chromatography or ultrafiltration on an instrument "Amicon" with thefilter XM-100.

The above-described process for producing urokinase derivatives isillustrated by the following scheme. ##STR5##

Gel-chromatographic recovery of the obtained derivatives in a columnpacked with Sephadex G-100 has shown an increase of the molecular massof the product (360-440 thousand D) as compared to native urokinase(31,000-54,000D). This points to the covalent addition of urokinase tofibrinogen (the product was recorded both through the protein (280 nm,spectrophotometry) and through the esterase catalytical activity ofurokinase).

At the same time, the comparison of properties of the resultingderivatives (Tables 1, 2 and 4) has shown that urokinase directlyattached to fibrinogen (without a diamine "leg", compound 5) isdistinguished, by a number of features, from derivatives of the enzymeattached to the vehicle matrix by means of a diamine.

Thus, the trends in variation of K_(M) and k_(cat), as shownhereinabove, point to an enhanced steric influence of the vehicle oncatalytical properties of urokinase. A higher thermal stability of theproduct (compound 5) proves a more rigid attachment of urokinase tofibrinogen in this case. The fibrinolytic activity of compound 5 in thiscase is lower than in compounds 1-4 which manifests a limited mobilityof the enzyme in this situation. The above-mentioned facts prove thatthe addition of urokinase to fibrinogen in compounds 1 to 4 is effectedthrough the "leg" of the aliphatic diamine.

The obtained urokinase derivatives after lyophilization comprise a whitefloccular powder readily soluble in water.

The resulting derivatives of urokinase according to the presentinvention have a molecular weight of 360,000-440,000D, proteincontent--10-30% by mass, esterase catalytic activity--30-60%.

The synthesis of urokinase derivatives covalently bonded with fibrinogenthrough an aliphatic diamine makes it possible to produce derivatives ofthe enzyme possessing a high thrombolytic activity, an increasedstability and affinity towards the thrombus material. Such derivativesof urokinase comprise effective agents for a systemic thrombolysis of aprolonged effect.

For a better understanding of the present invention the followingexamples of urokinase derivatives and procedures of their preparationare given hereinbelow by way of illustration.

EXAMPLE 1

7.83 mg of fibrinogen are dissolved in 6 ml of distilled water (3.8×10⁻⁶M) at room temperature, the solution is acidified by means of HCl to thepH=3.8 and then 4 mg of a carbodiimide are added thereto. 10 minutesthereafter, when the solution pH reaches its maximum value (pH=4.5) 2 mlof the solution of activated fibrinogen are taken and added at thetemperature of 4° C. to 2 ml of a solution of1,12-dodecamethylenediamine (3.8×10⁻⁴ M) in a 0.1M phosphate buffer(pH=8.3) and the mixture is incubated for 5 hours. Thereafter theincubation mixture is subjected to dialysis for 2 hours at a lowtemperature first against distilled water and then, under the sameconditions, against a 0.02M phosphate buffer at the pH=8.3 (product I).

The preparation of urokinase (24,000 IU) is dissolved in 2 ml ofdistilled water and dialyzed at a low temperature for 2 hours againstdistilled water and then for additional 2 hours--against a 0.05Mphosphate buffer with the pH=8.3. The dialyzed solution of urokinase isadded with 4 mg of carbodiimide and the mixture is incubated at a lowtemperature under stirring for 20 minutes (product II).

Thereafter, the solution of activated urokinase (product II) is addedwith 2 ml of a solution of fibrinogen-1,12-dodecamethylenediamine(product I). The mixture is incubated for 18 hours at the temperature of4° C., whereafter the resulting preparation is recovered from thereaction mixture by means of ultrafiltration on an "Amicon" instrumentwith the filter XM-100. A product is thus obtained which comprisesurokinase covalently bonded with fibrinogen by means of1,12-dodecamethylenediamine. The molecular mass is 440,000D, the contentof protein is 10% by mass, the retained esterase catalytic activity is50% as calculated for the starting urokinase.

EXAMPLE 2

The process is conducted in a manner similar to that described inExample 1, except that as the crosslinking agent1,10-decamethylenediamine is used.

A product is thus obtained which comprises urokinase covalently bondedwith fibrinogen by means of 1,10-decamethylenediamine. The molecularmass is 420,000D, the content of protein is 12% by mass, the retainedesterase catalytic activity is 45% as calculated for the startingurokinase.

EXAMPLE 3

The process is conducted in a manner similar to that described inExample 1, except that as the cross-linking agent1,7-heptamethylenediamine is used. A product is thus obtained whichcomprises urokinase covalently bonded with fibrinogen by means of1.7-heptamethylenediamine. The molecular mass is 400,000D, the contentof protein is 14% by mass, the retained catalytic esterase activity is30% as calculated for the starting urokinase.

EXAMPLE 4

The procedure of Example 1 hereinbefore is repeated, except that as across-linking agent 1,4-tetramethylenediamine is used. A product isobtained which comprises urokinase covalently bonded with fibrinogen bymeans of 1,4-tetramethylenediamine. The molecular mass is 385,000D, thecontent of protein is 15% by mass, the retained esterase catalyticactivity is 40% as calculated for the starting urokinase.

EXAMPLE 5

Urokinase is added to fibrinogen directly without any lengthening bridgeof an aliphatic diamine.

2 ml of urokinase (12,000 IU/ml) are dialyzed for 2 hours at thetemperature of 4° C. against distilled water and then against a 0.02Mphosphate buffer with the pH of 8.3. The solution of urokinase is addedwith 2 ml of fibrinogen activated with carbodiimide (3.8×10⁻⁶ M) and 1ml of a 0.1M phosphate buffer with the pH=8.3. The mixture is incubatedfor 18 hours at the temperature of 4° C. The resulting derivative isrecovered by ultrafiltration to give a product comprising urokinasecovalently bonded with fibrinogen. The molecular mass is 360,000D, thecontent of protein is 30% by mass the retained esterase activity is 32%as calculated for the starting urokinase.

The urokinase derivatives according to the present invention featuringan increased affinity to fibrin and a protracted fibrinolytic effect canbe used in medicine for the treatment of myocardial infarction,thromboses of coronary vessels, emboliae of lung arteries, thromboses ofbrain vessels and deep veins of limbs, and in a number of other cases.

We claim:
 1. Urokinase derivatives comprising urokinase covalentlybonded with fibrinogen or urokinase covalently bonded, through analiphatic diamine, with fibrinogen of the general formula: ##STR6##wherein P is fibrinogen, E is urokinase, R is either a single bond orstands for ##STR7## wherein n is 1-12, with a molecular mass of 360,000to 440,000D, a content of protein of 10-30% by mass, and an esterasecatalytic activity of 30-60%.
 2. The urokinase derivative of claim 1wherein said aliphatic diamine is 1,12-dodecamethylenediamine.
 3. Theurokinase derivative of claim 1 wherein said aliphatic diamine is1,10-decamethylenediamine.
 4. The urokinase derivative of claim 1wherein said aliphatic diamine is 1,7-heptamethylenediamine.
 5. Theurokinase derivative of claim 1 wherein said aliphatic diamine is1,4-tetramethylenediamine.
 6. The urokinase derivative of claim 1wherein R represents a single bond.
 7. A process for the preparation ofa urokinase derivative of the formula: ##STR8## which comprises reactinga compound of the formula: ##STR9## with a compound of the formula:##STR10## wherein P is fibrinogen, E is urokinase, R represents a singlebond or ##STR11## R₁ is C₂ H₅, R₂ is ##STR12## and n is 1-12, andrecovering said derivative having a molecular mass of 360,000 to440,000D, a protein content of 10-30% by mass, and an esterase catalyticactivity of 30-60%.